Theme 4: Replica.on
12.1 DNA Replica/on
• To say that the strands are an.parallel means that the 3ʹ hydroxyl of any
deoxyribose sugar in the backbone of one strand is opposite the 5ʹ
phosphate group of the corresponding deoxyribose sugar in the
backbone across the way
12.1.1 During DNA replica/on, the parental strands separate
and new partners are made.
• Watson and Crick also postulated that the pairing of DNA bases (A to T
and C to G) suggests a copying mechanism for gene.c material
◦ They suggested the two strands of parental duplex separate and
each serves as a model (i.e. template strand) for the synthesis of a
daughter strand which would have complementary base pairs to
that of the parents (i.e. 5'-ATGC-3' specifies 3'-TACG-5')
◦ Predicted semiconserva/ve replica/on (i.e. each new daughter
strand possesses one strand from the parent)
• Though this was debated with the conserva.ve model of
replica.on that was prevalent at the .me
How is DNA Replicated?
• Background- Watson and Crick discovery of structure of DNA in 1953
suggested a mechanism by which DNA is replicated; Experimental
evidence came with research by Meselson and Stahl in 1958
• Hypothesis- DNA replicates in a semiconserva.ve manner
• Alterna.ve Hypothesis- DNA replicates in a conserva.ve manner (i.e.
One molecule consists of two parental strands and the other consists of
two newly synthesized strands)
• Method- Meselson and Stahl dis.nguished parental strands from newly
synthesized strands using two isotopes of nitrogen
◦ "Old" strands were labeled with 15N and "new" strands were
labeled with 14N (which is the normal, lighter form of nitrogen)
, • Experiment-
◦ Density of DNA was measured by spinning it in a high-speed
centrifuge in tubes containing a solu.on of cesium chloride
• Predic.on- If DNA is replicated conserva.vely, half of the DNA in the
cells should be made of two heavy parental strands containing 15N and
half should be composed of two lighter daughter strands; If DNA is
replicated semi-conserva.vely, the daughter DNA molecules should
each consists of one heavy strand and one light strand
• Results- When the fully 15N-labeled parental DNA was spun, it
concentrated in a single heavy band with a density of 1.722 gm/cm3;
A`er one round of replica.on, the DNA formed a band at a density of
1.715 gm/cm3, which is the density expected of a duplex molecule
containing one heavy strand and one light strand (DNA composed only
of 14N would have a density of 1.708 gm/cm3)
◦ A`er two rounds of replica.on, half the molecules exhibited a
density of 1.715 gm/cm3, indica.ng a duplex molecule with one
heavy strand and one light strand, and the other half exhibited a
density of 1.708 gm/cm3, indica.ng a duplex molecule containing
two light strands
• Conclusion- DNA replicates semi-conserva.vely
• Meselson-Stahl experiment le` open the possibility that DNA replica.on
in eukaryotes might be different
◦ Methods for labeling DNA with fluorescent nucleo.des allowed
researchers to visualize en.re strands of eukaryo.c DNA and
follow each strand through replica.on
12.1.2 New DNA strands grow by the addi/on of nucleo/des
to the 3' end.
• Both daughter strands grow in length by the addi.on of nucleo.des
near the site where the parental strands separate (i.e. the replica/on
fork which is in the origin of replica/on)
◦ The new daughter strands have opposite orienta.ons, so that
near the replica.on fork the daughter strand in the bodom duplex
terminates in a 3ʹ hydroxyl, whereas that in the top duplex
terminates in a 5ʹ phosphate
12.1 DNA Replica/on
• To say that the strands are an.parallel means that the 3ʹ hydroxyl of any
deoxyribose sugar in the backbone of one strand is opposite the 5ʹ
phosphate group of the corresponding deoxyribose sugar in the
backbone across the way
12.1.1 During DNA replica/on, the parental strands separate
and new partners are made.
• Watson and Crick also postulated that the pairing of DNA bases (A to T
and C to G) suggests a copying mechanism for gene.c material
◦ They suggested the two strands of parental duplex separate and
each serves as a model (i.e. template strand) for the synthesis of a
daughter strand which would have complementary base pairs to
that of the parents (i.e. 5'-ATGC-3' specifies 3'-TACG-5')
◦ Predicted semiconserva/ve replica/on (i.e. each new daughter
strand possesses one strand from the parent)
• Though this was debated with the conserva.ve model of
replica.on that was prevalent at the .me
How is DNA Replicated?
• Background- Watson and Crick discovery of structure of DNA in 1953
suggested a mechanism by which DNA is replicated; Experimental
evidence came with research by Meselson and Stahl in 1958
• Hypothesis- DNA replicates in a semiconserva.ve manner
• Alterna.ve Hypothesis- DNA replicates in a conserva.ve manner (i.e.
One molecule consists of two parental strands and the other consists of
two newly synthesized strands)
• Method- Meselson and Stahl dis.nguished parental strands from newly
synthesized strands using two isotopes of nitrogen
◦ "Old" strands were labeled with 15N and "new" strands were
labeled with 14N (which is the normal, lighter form of nitrogen)
, • Experiment-
◦ Density of DNA was measured by spinning it in a high-speed
centrifuge in tubes containing a solu.on of cesium chloride
• Predic.on- If DNA is replicated conserva.vely, half of the DNA in the
cells should be made of two heavy parental strands containing 15N and
half should be composed of two lighter daughter strands; If DNA is
replicated semi-conserva.vely, the daughter DNA molecules should
each consists of one heavy strand and one light strand
• Results- When the fully 15N-labeled parental DNA was spun, it
concentrated in a single heavy band with a density of 1.722 gm/cm3;
A`er one round of replica.on, the DNA formed a band at a density of
1.715 gm/cm3, which is the density expected of a duplex molecule
containing one heavy strand and one light strand (DNA composed only
of 14N would have a density of 1.708 gm/cm3)
◦ A`er two rounds of replica.on, half the molecules exhibited a
density of 1.715 gm/cm3, indica.ng a duplex molecule with one
heavy strand and one light strand, and the other half exhibited a
density of 1.708 gm/cm3, indica.ng a duplex molecule containing
two light strands
• Conclusion- DNA replicates semi-conserva.vely
• Meselson-Stahl experiment le` open the possibility that DNA replica.on
in eukaryotes might be different
◦ Methods for labeling DNA with fluorescent nucleo.des allowed
researchers to visualize en.re strands of eukaryo.c DNA and
follow each strand through replica.on
12.1.2 New DNA strands grow by the addi/on of nucleo/des
to the 3' end.
• Both daughter strands grow in length by the addi.on of nucleo.des
near the site where the parental strands separate (i.e. the replica/on
fork which is in the origin of replica/on)
◦ The new daughter strands have opposite orienta.ons, so that
near the replica.on fork the daughter strand in the bodom duplex
terminates in a 3ʹ hydroxyl, whereas that in the top duplex
terminates in a 5ʹ phosphate
